The mutational spectrum in whole exon of p53 in oral squamous cell carcinoma and its clinical implications

Mutations in p53 are common in human oral squamous cell carcinoma (OSCC). However, in previous analyses, only detection of mutant p53 protein using immunohistochemistry or mutations in some exons have been examined. Full length mutant p53 protein in many cases shows a loss of tumor suppressor function, but in some cases possibly shows a gain of oncogenic function. In this study, we investigate relationships of outcomes with the mutational spectrum of p53 (missense and truncation mutations) in whole exon in OSCC. Specimens from biopsy or surgery (67 cases) were evaluated using next-generation sequencing for p53, and other oncogenic driver genes. The data were compared with overall survival (OS) and disease-free survival (DFS) using univariate and multivariate analyses. p53 mutations were detected in 54 patients (80.6%), 33 missense mutations and 24 truncation mutations. p53 mutations were common in the DNA-binding domain (43/52) and many were missense mutations (31/43). Mutations in other regions were mostly p53 truncation mutations. We detected some mutations in 6 oncogenic driver genes on 67 OSCC, 25 in NOTCH1, 14 in CDKN2A, 5 in PIK3CA, 3 in FBXW7, 3 in HRAS, and 1 in BRAF. However, there was no associations of the p53 mutational spectrum with mutations of oncogenic driver genes in OSCC. A comparison of cases with p53 mutations (missense or truncation) with wild-type p53 cases showed a significant difference in lymph node metastasis. DFS was significantly poorer in cases with p53 truncation mutations. Cases with p53 truncation mutations increased malignancy. In contrast, significant differences were not found between cases with p53 missense mutations and other mutations. The p53 missense mutation cases might include cases with mostly similar function to that of the wild-type, cases with loss of function, and cases with various degrees of gain of oncogenic function.

Immunohistochemical staining. Using specimens resected in biopsy or surgery, immunohistochemical staining was performed for p53 and p16. Resected tissue was immediately fixed with 10% neutral-buffered formalin solution and paraffin-embedded to prepare 4-μm thin sections. The sections were deparaffinized with xylene and serially rehydrated with ethanol. Antigen was activated by microwave at 95 °C for 10 min (pH 6.0 citrate buffer solution), washed with phosphate-buffered saline (PBS), and then treated with 0.3% hydrogen peroxide in methanol for 20 min for inhibition of endogenous peroxidase; a 30-min total blocking time. X0909 Protein Block Serum-Free (Dako, Glostrup, Denmark) was used for blocking. Incubations with mouse antihuman p53 monoclonal antibody (Clone DO-7, 1:50 dilution, Dako) and mouse anti-human p16 monoclonal antibody (Clone G175-405, 1:200 dilution, BD Pharmingen, San Diego, CA) as primary antibodies were performed for 60 min. Thereafter, the procedure followed the polymer-immune complex method using Envision (K4001, Dako). In p53 and p16 immunostaining, a stain coinciding with a tumor cell nucleus was regarded as positive 24,31 . Samples were evaluated by TH (Toshiki Hyodo) and HK using measurements in ≥ 5 visual fields at 200 × magnification: cases with no positive findings were regarded as negative, and those with a positive finding in one or more visual fields were regarded as positive.
DNA extraction. From each patient with OSCC, genomic DNA (gDNA) was extracted from an approximately 25-mg specimen from the tumor center in the biopsied or surgically excised material using a QIAamp Fast DNA Tissue Kit (Qiagen, Hilden, Germany) 7,32 . About 100 ng/7.5 µl of gDNA was used for the next-generation sequencing (NGS). Histological analysis was performed on the remaining specimen to confirm the presence of active tumor cells.
Procedure for NGS. A  Statistical analysis. Cases with p53 mutation and wild-type cases were compared by Chi-square test, with p < 0. 05 regarded as significant. In univariate and multivariate analyses, age, gender, smoking, drinking, UICC TNM classification, Y-K mode of invasion, p53 mutational spectrum (missense mutation, truncation mutation, wild-type/p16 status) were included as potential risk factors. Hazard ratios (HR) were calculated in a Cox proportional hazard model, again with p < 0. 05 regarded as significant. A two-sided 95% confidence interval was

Results
Characteristics of the patients and the p53 mutational spectrum. Sixty-seven patients with primary OSCC including 39 males (58.2%) and 28 females (41.8%) who underwent radical tumor resection were enrolled in this study (  19. On the Y-K mode of invasion, Y-K-1 case was 9, Y-K-2 case was 14, Y-K-3 case was 28, Y-K-4C case was 15, and Y-K-4D case was 1. p53 mutational spectrum. p53 mutation was found in 54 of 67 patients (80.6%), and a total of 57 mutations were detected, including 3 cases with double mutations (Fig. 1). These included 33 missense mutations and 24 truncation mutations (nonsense mutation 10, frame-shift variant 9, splice-site variant 3, in-frame deletion 2). In the wild-type cases, immunohistology showed that 6 and 7 patients were p16-positive (oncogenic HPV was infected and integrated) and-negative (oncogenic HPV was not infected), respectively. For verification of the p53 mutations, the immunohistological and NGS results were evaluated in combination. No abnormal accumulation of p53 protein in the nucleus was observed immunohistologically in cases in which NGS indicated no mutation ( Fig. 2A,D). In contrast, cases in which a mutation was identified by NGS had abnormal nuclear accumulation of p53 protein immunohistologically (Fig. 2B,E). In cases in which mutation was unclear on NGS despite observation of p53 protein accumulation in the nucleus, the read depth decreased from 500 to 200 and the presence of a mutation was confirmed by reinvestigation. In cases with a p53 truncation mutation detected by NGS, the absence of abnormal p53 protein accumulation in the nucleus was confirmed immunohistologically (Fig. 2C,F).
p53 mutational landscape in OSCC. The p53 mutational spectrum in OSCC showed a diverse distribution of mutations (Fig. 3). However, mutations were common in the DBD (43/52) and many of these were missense mutations (31/43). Some truncation mutations were also present in the DBD (13/43). Mutations in other regions, such as the transactivation domain (TAD), proline-rich domain (PRD), and oligomerization domain (OD) (7/9), were mostly truncation mutations, with missense mutations found in only 2 cases.

Figure 2.
Immunohistological evaluation of p53 in oral squamous cell carcinoma (OSCC) cases. No abnormal accumulation of p53 protein in the nucleus was observed in cases in which no mutation was found by nextgeneration sequencing (NGS) (A,D). In cases with a mutation detected by NGS, abnormal nuclear accumulation of p53 protein was found immunohistologically (B,E). In cases with a truncation mutation detected by NGS, the absence of abnormal nuclear accumulation of p53 protein was shown immunohistologically (C,F).  Characteristics of the patients and the p53 mutational spectrum (statistical analysis). There were no significant differences in the clinical and biological characteristics among cases with a p53 missense mutation, p53 truncation mutation, wild-type p53 and p16-positive, and wild-type p53 and p16-negative status (data not shown). However, a comparison of cases with p53 mutations (missense or truncation) with all wildtype p53 cases showed a significant difference in pN (p = 0.005) and Y-K mode of invasion (p = 0.021) by Chisquare test (Table 4). Risk factors for OS and DFS were investigated based on clinical and biological characteristics using binominal logistic regression univariate and multivariate analyses (Tables 5, 6). pN was a significant poor prognostic factor for OS in both analyses (univariate: p = 0.002, multivariate: p = 0.005) as well as for DFS in both analyses (univariate: p = 0.005, multivariate: p = 0.026). Y-K mode of invasion was associated with DFS in univariate analysis (p = 0.042). DFS also differed significantly in cases with truncation mutations compared with all other cases (missense mutations and wild-type p16-positive or negative) in univariate analysis (p = 0.050).

Discussion
In this study, p53 mutation was found in 54 of 67 patients (80.6%), and a total of 57 mutations were detected, including 3 cases with double mutation. The p53 mutation site was diverse, but mostly found in the DBD, and many mutations in the DBD were missense mutations. These findings for OSCC are similar to those in previous reports for other tumors 33,34 . Olivier et al. found occasional missense mutations throughout the coding region, but 97% of these mutations were detected in the exon coding for the DBD 35 . Missense mutation-induced amino acid substitution in the DBD changes DNA binding capacity and leads to a loss or change in transcriptional activation 36 . In the DBD, 6 major hotspots have been identified at codons 175, 245, 248, 249, 273, and 282 37,38 .
In the current study, mutations were detected at all of these hotspots, codons 175 (3 cases), 245 (1 case), 248 (3 cases), 249 (1 case), 273 (1 case), and 282 (2 cases), respectively. Surprisingly, in regions other than the DBD, truncation mutations were found in many cases, but only 2 missense mutations. These 2 missense mutations were likely to have been single nucleotide polymorphisms (SNP) based on the allele frequency, and thus had little biological significance. Our investigation of associations of the p53 mutational spectrum with mutations of oncogenic driver genes in OSCC did not produce any significant results. Chaudhary performed a comparison of the mutation rate of driver genes in head and neck SCC between African-Americans and Caucasians, and found high frequencies of p53 and HRAS mutations 39 . In our results, the mutation rates of p53 and NOTCH1 were high, but that for PIK3CA was low. These results might reflect the characteristics of the driver genes in Asians, but there are no previous data for comparison. There were 7 cases of wild-type p53 (p16-negative) and an activating mutation of HRAS was found in 2 of these cases (codon 13 G13R and G13V). Mutation of HRAS was noted in only one (codon 12 G12S) of 31 cases with missense mutations, and there was no HRAS mutation in cases with truncation mutations. Mutation of NOTCH1 was more frequent in wild-type p53 cases than in mutant p53 cases (including missense www.nature.com/scientificreports/ and truncation mutations). Although there was no significant association of mutations of HRAS and NOTCH1 with the p53 mutational spectrum, these findings may indicate that mutation of other major signaling pathways occurs at a high frequency in cancer cells with normal p53 function. Cases with p53 mutation had significantly higher rates of pathological lymph node metastasis-positive status and Y-K mode of invasion ≥ 3, compared to wild-type cases. OS and DFS were significantly shorter in cases with lymph node metastasis, and DFS was significantly worsened by greater Y-K mode of invasion. Cases with p53 truncation mutations also had a significantly shorter DFS. Use of the Kaplan-Meier method with a log-rank test showed no significant differences in OS or DFS among the 4 types in the p53 mutational spectrum. However, a truncation mutation was a significant poor prognostic factor for DFS, but a missense mutation was not found as a prognostic factor for OS or DFS. Singh et al. reported p53 mutational spectrum and its role in prognosis of oral cancer patients from India 40 . They mentioned that OS and DFS of OSCC patients with p53 truncation mutation and transcriptionally non-active mutations were significantly lower than those with wild-type p53. www.nature.com/scientificreports/ These results were consistent with the results from our Japanese data. They examined 46 patients with OSCC and most of the patients (86.9%) were tobacco user (smoking and chewing). Although the tobacco habit in India was quite different from that in Japan, tobacco smoking and alcohol drinking was not related with the p53 mutational spectrum in our study. These findings suggest that p53 function was completely lost in cases with truncation mutations, which increased malignancy. This may explain the significant differences between truncation and non-truncation mutation cases. In contrast, significant differences were not found between cases with p53 missense mutations and other mutations in this study. This may have been due to the p53 missense mutation cases being a mixture of those with mostly similar function to that of the wild-type, cases which loss of function, and cases with various degrees of gain of oncogenic function. We are planning to investigate possible acquiring functions based on the missense mutation site and pattern; i.e., a 'oncogenic mutation of p53' , by preparing a database and identifying There are about 200 amino acids in the DBD and about 20 kinds of amino acids can be substituted by mutation. Therefore, there are about 4,000 patterns of single amino acid substitutions. There were few cases with multiple mutations in the DBD in our study or in previous reports. These findings suggested that a single amino acid  www.nature.com/scientificreports/ substitution was enough to change p53 protein function and no subsequent changes were required. Therefore, to prepare a database of functional changes corresponding to a mutation in the DBD, a search for a maximum of 4,000 mutants is sufficient. When we start a search for the clinical outcomes (invasive and metastatic potentials, and prognosis) as a result of functional abnormality (loss or gain of function) of p53 in each case with known p53 mutational site, it may enable realistic data accumulation. Separately, a search for functional analysis mutated p53 products in vitro may help the construction of the database. We are planning to conduct the experiment and reported the preliminary results, in which transcriptional activity of mutated p53 products for known target genes 26,27 or for new target genes, or the ability to bind to p63 and p73 as a dominant negative effect are examined. By completing these databases, genome diagnosis based on the p53 mutational spectrum may become possible and will provide important information for decision-making with regard to the treatment strategy for OSCC. Neskey et al. proposed an evolutionary action score for p53 protein to stratify the tumors harboring p53 mutation as high or low risk by computational approach 41 . They also validated this system in in vitro and in vivo. Patients with high risk p53 mutations had poorest survival outcomes and shortest time to the development of distant metastasis. Tumor cells expressing high risk p53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastasis. Although our approach was somewhat different from their approach, the concept and the goal might be similar to those in their approach. Furthermore, Phase II clinical trial (ECOG-ACRIN 3132) in head and neck cancer harboring several patterns of p53 gene (disruptive or nondisruptive p53 mutation, p53 wild-type) are ongoing 42 . We are also planning to conduct a clinical trial to select a treatment intensity (post-surgical chemo-radiation therapy and/or molecular targeted therapy) based on the p53 mutational spectrum database.

Data availability
The data that support the findings of our study are available from the corresponding author upon reasonable request. Nucleotide sequence data reported are available in the DDBJ Sequenced Read Archive under the accession numbers DRA014726. https:// ddbj. nig. ac. jp/ resou rce/ sra-submi ssion/ DRA01 4726.